不成熟CD8α^+ DC经同种白血病抗原冲击后移植物抗白血病效应的体外研究  

Graft-versus-leukemia effect induced by impulsing with allogeneic leukemia antigen plus immature CD8α^+ dendritic cells in vitro

作　　者：徐霖[1] 纳宁[2] 陈康[1] 曹开源[3] 袁广卿[3] 傅强[3] 朱蓓莉 

机构地区：[1]中山大学中山医学院免疫学教研室,广东广州510080 [2]中山大学附属第三医院肾移植科,广东广州510630 [3]中山大学中山医学院微生物学教研室,广东广州510080 [4]东莞市卫生学校,广东东莞523000

出　　处：《中国病理生理杂志》2011年第10期2005-2009,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81000230);广东省自然科学基金资助项目(No.8451008901000213No.07001683);广东省科技计划资助项目(No.2010B031600052)

摘　　要：目的:观察不成熟CD8α+树突状细胞(DC)体外经同种异基因白血病细胞全抗原冲击后的移植物抗白血病(GVL)效应。方法:C57BL/6(H-2b)小鼠的骨髓细胞以GM-CSF+IL-4+SCF+Flt3L体外诱导不成熟CD8α+DC,第3 d加入0 mg/L、2.5 mg/L、5 mg/L、10 mg/L和20 mg/L BALB/c(H-2d)小鼠来源的同种异基因EL9611红白血病细胞抗原冲击,冲击后DC与同系(H-2b)T细胞按DC/T 1∶1、2∶1、4∶1比例共培养,MTT法观察T细胞增殖情况,ELISA法检测上清中IFN-γ和IL-10的含量,LDH释放法检测T细胞对EL9611细胞的杀伤活性。以成熟DC为对照,观察同种异基因不成熟CD8α+DC体外对EL9611白血病细胞的GVL效应。结果:同种红白血病全抗原冲击后不成熟CD8α+DC可有效刺激同系T细胞增殖,作用随DC/T比例增加而增加,增殖效应在小剂量抗原(≤5 mg/L)冲击时更明显(P<0.05);Ag冲击后不成熟CD8α+DC刺激T细胞分泌IFN-γ和IL-10明显增高(P<0.05),IL-10的分泌与不成熟CD8α+DC发挥GVL效应存在一定的负相关关系,Ag冲击浓度较低而IL-10分泌少时,T细胞增殖活性较强。杀伤实验结果显示,白血病特异性T细胞对EL9611靶细胞的杀伤活性随抗原冲击浓度的增加而升高,浓度为2.5 mg/L时,杀伤率即可达90%,非特异性杀伤实验显示白血病特异性T细胞对同种异基因正常脾细胞的杀伤活性低于对照组(P<0.05),表明杀伤作用为抗原特异性杀伤,对正常细胞无明显杀伤。结论:经同种异基因白血病抗原冲击后不成熟CD8α+DC体外能刺激同系T细胞产生一定的GVL效应。AIM：To observe the graft-versus-leukemia（GVL） effect of immature CD8α＋ dendritic cells（DC） plus impulsing of allogeneic leukemia whole-cell antigen in vitro.METHODS： Bone marrow cells of C57BL/6（H-2b） mice were induced by GM-CSF ＋IL-4 ＋SCF ＋Flt3L in vitro to prepare immature CD8α＋DC.On day 3,different doses of BALB/c（H-2d）-derived allogeneic EL9611 leukemia antigen at concentration series of 0 mg/L,2.5 mg/L,5 mg/L,10 mg/L and 20 mg/L were added.After impulsing,DCs were co-cultured with syngeneic T cells at DC/T ratio of 1∶ 1,2∶ 1 or 4∶ 1.Proliferation of the T cells was measured by MTT method.The concentrations of IFN-γ and IL-10 in cultured supernatants were detected by ELISA.Cytotoxicity of T cells on EL9611 cells was measured by LDH releasing assay.Mature DC served as the control and in vitro GVL effect of allogeneic immature CD8α＋DC on EL9611 leukemia cells was observed.RESULTS： Immature CD8α＋DC plus impulsing of allogeneic leukemia antigen effectively stimulated syngeneic T cell proliferation,which increased with DC/T ratio.The effect of proliferation was more obviously,especially in the presence of antigen at low dose（≤5 mg/L,P〈0.05）.The secretions of IFN-γ and IL-10 in the T cells stimulated with antigen plus immature CD8α＋DC became higher significantly（P〈0.05）.There was a certain negative correlation between the secretion of IL-10 and the GVL effect of CD8α＋ immature DC.When the concentration of impulsing antigen and IL-10 secretion was low,T cell proliferation became higher.The results of cytotoxicity assay showed that the cytotoxicity activity of leukemia specific T cells on EL9611 target cells increased with the antigen concentration,and the killing rate reached 90% when the concentration of the antigen was 2.5 mg/L.Non-specific cytotoxicity assay showed that cytotoxicity activity of leukemia specific T cells on allogeneic normal spleen cells was lower than that in control group（P〈0.05）,indicating that it was a specific killing,a

关 键 词：移植物抗白血病 树突细胞 T淋巴细胞 

分 类 号：R363[医药卫生—病理学]